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Food Hydrocolloids 23 (2009) 886–895

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Food Hydrocolloids

journal homepage: www.elsevier .com/locate/ foodhyd

Concurrent phase separation and gelation in mixed oat b-glucans/sodiumcaseinate and oat b-glucans/pullulan aqueous dispersions

A. Lazaridou*, C.G. BiliaderisLaboratory of Food Chemistry & Biochemistry, Department of Food Science and Technology, School of Agriculture, Aristotle University, P.O. Box 256, Thessaloniki 541 24, Greece

a r t i c l e i n f o

Article history:Received 17 March 2008Accepted 22 May 2008

Keywords:Oat (1/3), (1/4)-b-D-glucanSodium caseinatePullulanPhase separationGelationMolecular weight

* Corresponding author. Tel.: þ30 2310 991716; faxE-mail address: athlazar@agro.auth.gr (A. Lazarido

0268-005X/$ – see front matter � 2008 Elsevier Ltd.doi:10.1016/j.foodhyd.2008.05.008

a b s t r a c t

The phase separation behavior of mixed oat b-glucans/sodium caseinate and oat b-glucans/pullulanaqueous dispersions at 20 �C has been studied. The concentration of b-glucans required for induction ofphase separation and the physical state of the separated phases, as revealed by visual observations anddynamic rheometry, depended on the molecular weight of b-glucans and the initial polymeric compo-sition. For b-glucans with apparent molecular weights (Mw) 35 and 65� 103 the b-glucan concentrationat which thermodynamic incompatibility occurred decreased from about 2–2.5% (w/w) at low concen-trations (w0.2%) of sodium caseinate or pullulan to about 1–1.5% (w/w) b-glucans at high levels (up to7.5% w/w) of the second biopolymer; these bi-phasic systems consisted of an upper liquid phase anda lower gel-like phase. For b-glucans with Mw of 110� 103, a bi-phasic system with two liquid phasesappeared above a certain b-glucan concentration, which decreased from approximately 4% to 1% (w/w)with increasing sodium caseinate levels in the range of 0.2–7.5% (w/w). With further increase in b-glucanconcentration, the lower phase turned into a gel, and at even higher b-glucan concentrations, thepolymer demixing process was ‘arrested’ by chain aggregation events, leading to a macroscopically singlegel phase. Generally, the aggregation of b-glucans seemed to interfere with the phase separationphenomenon resulting in an increase of b-glucan concentration in the lower phase between 5% and 110%and only a slight increase of sodium caseinate or pullulan concentration in the upper phase (<10%), dueto kinetic entrapment of the polymeric components into a highly viscous medium.

� 2008 Elsevier Ltd. All rights reserved.

1. Introduction

The (1/3), (1/4) b-D-glucans (b-glucans) are linear homo-polysaccharides of consecutively linked (1/4)-b-D-glucosyl resi-dues (i.e. oligomeric cellulose segments) that are separated bysingle (1/3)-linkages. The most important sources of these poly-saccharides are oat and barley. b-Glucans display all the beneficialfunctional properties of viscous and gel-forming food hydrocolloidscombined with all the physiological properties of dietary fibres. Thephysical properties of b-glucans, such as solubility and rheologicalbehavior in the solution and gel states, and thus their technologicalfunctionality are controlled by their molecular features, such as thedistribution of cellulosic oligomers, the linkage pattern and theirmolecular weight as well as by temperature and polysaccharideconcentration (Lazaridou & Biliaderis, 2007). Cereal b-glucans ex-hibit health benefits, such as reducing blood serum cholesterol andregulating postprandial blood glucose and insulin levels (Wood,2002). The physiological effects of b-glucans are also related to theconcentration and molecular weight of the solubilized b-glucans in

: þ30 2310 991797.u).

All rights reserved.

the gastro-intestinal tract, mainly reflected in the viscosity profileof these biopolymers.

Sodium caseinate is a milk protein product, used as functionalingredient in many foods, such as dairy desserts. The proteincomponents in sodium caseinate preparations have lost theirmicellar organization (Kinsella, 1984). Pullulan is a linear mixed-linkage a-D-glucan, consisting mainly of maltotriose units inter-connected via a-(1/6) linkages. This polysaccharide is a typicalflexible random coil polymer, giving solutions of relatively lowviscosity with almost Newtonian flow behavior (Rees, 1977).

Interactions between different hydrocolloids in food systemsare important determinants for the macroscopic properties ofproducts, including flow behavior, stability, texture and mouth feel(de Kruif & Tuinier, 2001). Immiscibility between unlike polymersin mixtures is a well-known phenomenon and generally leads tophase separation, at which each phase is being enriched in one ofthe polymers (Tolstoguzov, 2003). However, there are some pro-cesses, such as aggregation of polymer chains that can interferewith the phase separation phenomenon and thus, influence thefunctional properties and stability of the food matrix. There area number of studies describing the relations between phase sep-aration and gelation phenomena in aqueous mixtures of twohydrocolloids (Alevisopoulos, Kasapis, & Abeysekera, 1996; Alves,

A. Lazaridou, C.G. Biliaderis / Food Hydrocolloids 23 (2009) 886–895 887

Antonov, & Goncalves, 1999, 2000; de Bont, Luengo Hendriks, vanKempen, & Vreeker, 2004; Bourriot, Garnier, & Doublier, 1999;Butler, 2002; Garnier, Schorsch, & Doublier, 1995; Kasapis, Morris,Norton, & Gidley, 1993; Loren & Hermansson, 2000; Loren et al.,2001; Loren, Langton, & Hermansson, 1999; Manoj, Kasapis, &Chronakis, 1996; Owen & Jones, 1998; Sciortino, Bansil, Stanley, &Alstrom, 1993; Tromp & Jones, 1996; Tromp, Rennie, & Jones,1995). In these systems, at least one of the hydrocolloids wasgelled and the phase separation is determined by macroscopic ormicroscopic observations. Enhanced viscosity or gelation of thesystem can influence phase separation kinetics by inducingrestricted mobility of the components, thus leading to non-equilibrium situations and kinetically entrapped states; this phe-nomenon that hinders macroscopic phase separation is known as‘arrested’ phase separation.

Recently, the potential interactions of cereal b-glucans withother ingredients into fermented dairy products have been in-vestigated (Kontogiorgos, Ritzoulis, Biliaderis, & Kasapis, 2006;Lazaridou, Vaikousi, & Biliaderis, 2008). Kontogiorgos et al. (2006)studied the microstructural and mechanical behavior of acid-setsodium caseinate/barley b-glucan mixtures. The resultant mixedgels were phase-separated at a microscopic level and were domi-nated by the protein component at low b-glucan concentrations(�3% w/w), whereas increasing amounts of the polysaccharide ledto its structural predominance. Inclusion of barley b-glucans inacid-set skimmed milk gels weakens the protein network, showingalso some evidence for incompatibility between these poly-saccharides and milk proteins (Lazaridou et al., 2008). Theseinteractions were found to largely depend on the concentration andmolecular structure of b-glucans.

The bulk phase separation behavior of mixed aqueous disper-sions of cereal b-glucans with other hydrocolloids under neutralconditions, to simulate fortified liquid products with these bio-active ingredients, has not been studied yet. In the present work,the phase changes of mixed oat b-glucan/sodium caseinate (pro-tein) or oat b-glucan/pullulan (a-D-glucan) dispersions have beenexplored.

2. Materials and methods

2.1. Materials and molecular characterization of polysaccharides

Three mixed-linkage (1/3), (1/4) b-D-glucan preparationsfrom oats (OGL110, OGL65 and OGL35) differing in molecularweight were used in this study. These samples were isolates fromoat flours or bran concentrates which were provided by CEBA(Lund, Sweden); the purification protocol of these preparations aswell as their molecular characterization were described in detailelsewhere (Lazaridou, Biliaderis, & Izydorczyk, 2003a). The appar-ent molecular weight of the b-glucan samples, obtained witha high-performance size exclusion chromatography (HPSEC) sys-tem combined with a refractive index detector (RI), were 110, 65and 35�103 Da, for OGL110, OGL65 and OGL35, respectively. TheirDP3/DP4 molar ratio, determined by lichenase treatment and high-performance anion-exchange chromatography (HPAEC) combinedwith a pulsed amperometric (PAD) detector, was found to be 2.3 forall b-glucan samples. The intrinsic or limiting viscosities, [h], ofaqueous solutions of OGL110, OGL65 and OGL35 b-glucans, mea-sured with Ubbelohde capillary viscometers at 20� 0.1 �C, werefound 1.85, 0.91 and 0.67 dl/g, respectively (Lazaridou et al., 2003a).In addition, the oat b-glucan preparations were characterized fortheir high b-glucan content (93–95% d.b.), and low protein (2–4%d.b.) levels, as assessed by the method of McCleary and Glennie-Holmes (1985), using the Megazyme� mixed-linkage b-glucanassay kit, and by the method of Lowry (Lowry, Rosebrough, Farr, &Randall, 1951), respectively.

The sodium caseinate (Na-CAS) was obtained from Wako PureChemical Industries Ltd. (Japan); its protein content amounts toapproximately 80%. Pullulan (PULL) was a food grade preparation,highly pure (>99%) from Hayashibara Biochemical Laboratory(Okayama, Japan), named PI20. The weight-average molecularweight (Mw) of the pullulan sample was calculated as 361�103 Dausing a high-performance size exclusion chromatography (HPSEC)system combined with refractive index (RI) and multiangle laserlight scattering (MALLS) detectors. The [h] value of aqueouspullulan solutions at 20� 0.1 �C was found to be 0.56 at20� 0.1 �C, as described by Lazaridou, Biliaderis, and Kontogiorgos(2003b).

2.2. Methods

Stock solutions of polysaccharides (oat b-glucans, 2.0–9.0% w/w,and pullulan, 0.5–12.0% w/w) were prepared in sealed vials bystirring the powders in distilled water until complete solubilizationof the material was achieved at 90 �C; the solutions were thencooled under running water at ambient temperature. Sodium ca-seinate stock solutions (1.5–13.5% w/w) were made by dispersingthe powder in distilled water under continuous stirring at roomtemperature, thus achieving solubilization; the solutions weredegassed under vacuum. The phase behavior of oat b-glucan/so-dium caseinate and oat b-glucans/pullulan systems was studied bypreparing a large number of binary mixtures differing in bio-polymer composition and b-glucan molecular weight. The corre-sponding stock preparations were mixed in appropriateproportions into 15 ml centrifuge tubes; homogenization was per-formed by vigorous shaking. The pH range of oat b-glucan/sodiumcaseinate dispersions was 6.3–6.4, whereas the pH of all oat b-glucans/pullulan aqueous dispersions was 6.2. The total volume ofthe mixtures in the tubes was 12 ml and aliquots of 1–2 ml weretaken for concentration determination of the individual hydrocol-loids in the initial mixtures and for the dynamic rheometrymeasurements.

The mixtures were stored in the tubes for 18 h at 20 �C andcentrifuged at 900g for 2 h. The phase separation boundary wasthen detected by visual observation since the two phases wereclearly separated when phase separation occurred. In the case ofphase separation, the volumes of the upper and lower phases ineach mixture were measured and expressed as a percentage of thetotal volume (% v/v) and then the two layers were carefullyseparated.

The composition of the initial mixtures used for the construc-tion of phase diagrams as well as the composition of the upper andlower phases was determined. b-Glucan concentration was mea-sured by the phenol-sulfuric method for total carbohydrates(Dubois, Gilles, Hamilton, Rebers, & Smith, 1956) using b-glucansolutions as standard, while the sodium caseinate concentrationwas determined using the method of Lowry (Lowry, Rosebrough,Farr, & Randall, 1951). For determination of pullulan concentration,samples were firstly digested by a purified pullulanase preparationfrom Klebsiella planticola (Megazyme) in an acetate buffer (0.1 M,pH 5.3) at 37 �C for 1 h and then pullulan concentration was de-termined by the Nelson–Somogyi method for reducing sugars(Nelson, 1944), using a pullulan hydrolyzate as a standard. In thecase of b-glucan/pullulan systems, the b-glucan concentration wascalculated by subtracting the pullulan concentration from the totalpolysaccharide concentration measured by the phenol-sulfuricmethod.

The state (liquid or gel) of the initial mixtures and the separatedphases after aging/centrifugation was determined by visual obser-vation of the flow of inverted tubes and further characterized by therespective mechanical spectra obtained by dynamic rheometry.Rheological measurements of the samples were performed by

0

1

2

3

4

5

6

OG

L35 (%

w

/w

)

c

0

1

2

3

4

5

6

0 2 4 6 8Na-CAS (% w/w)

OG

L110 (%

w

/w

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One gel phaseTwo phases (upper liquid; lowergel)Two liquid phasesOne liquid phase

a

0

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2

3

4

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/w

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Two phases (upper liquid; lower gel)One liquid phase

b

1 3 5 7

0 2 4 6 81 3 5 7

Na-CAS (% w/w)

0 2 4 6 81 3 5 7

Fig. 1. Bulk phase separation of oat b-glucans/sodium caseinate aqueous dispersionsafter centrifugation (900g� 2 h) following 18 h aging at 20 �C; mixtures denoted asone gel phase systems resulted after the aging process and without centrifugation. Allpoints represent the initial composition of the biopolymer mixtures. OGL110 andOGL35: oat b-glucans with apparent molecular weights of 110 and 35�103,respectively; Na-CAS: sodium caseinate.

A. Lazaridou, C.G. Biliaderis / Food Hydrocolloids 23 (2009) 886–895888

a rotational Physica MCR 300 rheometer (Physica MesstechnicGmbH, Stuttgart, Germany), using a parallel plate geometry(25 mm diameter and 1 mm gap); the temperature was regulatedby a Paar Physica circulating bath and a controlled peltier system(TEZ 150P/MCR) with an accuracy of �0.1 �C. Small deformationoscillatory measurements of G0 (storage modulus), G00 (loss modu-lus), and tan d (G00/G0) were performed with a strain of 0.1% anda range of frequencies (0.1–10 Hz) at 20 �C.

3. Results

Immediately after mixing of oat b-glucans with other hydrocol-loids such as sodium caseinate (protein) and pullulan (a-D-glucan)resulted in homogeneous liquid dispersions. However, after 18 haging at 20 �C and following centrifugation at 900g for 2 h, a clearmacroscopic phase separation occurred for some of the testedmixtures, showing incompatibility between b-glucans and theother two hydrocolloids.

In mixed oat b-glucans/sodium caseinate aqueous dispersions,the concentration of b-glucans at which mono-phasic or bi-phasicsystems after aging and centrifugation occurred as well as thephysical state of the one or two phases were found to depend onthe molecular weight of b-glucans and the initial polymeric com-position (Fig. 1). For oat b-glucan/sodium caseinate aqueous mix-tures containing the highest molecular weight b-glucan sample(OGL110), the phase (state) diagram exhibited four different zonesdepending on the composition of the mixtures (Fig. 1a). Up toa certain b-glucan concentration, varying with the sodium casein-ate concentration, no phase separation was observed, since forthese mixtures a single liquid phase was found. However, with anincrease of b-glucan concentration in the aqueous mixture, bulkphase separation appeared after aging and centrifugation; themixtures appeared to separate into two distinct liquid phases. Witha further increase in b-glucan concentration, there was a change inthe state of the lower phase, which developed into a gel-likematerial. At even higher b-glucan concentrations, above these bi-phasic areas, a mono-phasic zone was found, at which the mixturesafter the aging period (18 h at 20 �C) appeared as homogeneousgels without centrifugation. On the other hand, for the mixed oat b-glucan/sodium caseinate aqueous dispersions containing the twolower molecular weight b-glucan samples (OGL65 and OGL35), twoareas were observed on their phase (state) diagrams (Fig. 1b and c).Up to a certain b-glucan concentration, macroscopically no phaseseparation appeared after the aging and centrifugation process,while with further increase of b-glucan level in the mixturea phase-separated system was evident; an upper liquid phase anda lower gel phase.

The liquid- or gel-like state of initial mixtures and separatedphases were determined by macroscopic observations of invertedtubes containing these systems. A sample was described as liquidwhen inverting the tube there was a flowing liquid, even if it wasvery viscous; instead, a sample was characterized as gelling whenthe material was gravitationally stable in the inverted tube.Moreover, the rheological behavior of the initial mixtures and theirseparated phases was confirmed by the respective mechanicalspectra obtained by small strain oscillatory measurements, asshown in Fig. 2 for the oat b-glucans/sodium caseinate systems. Theinitial mixtures and the upper phases displayed the typical visco-elastic behavior of a macromolecular dispersion, where G00 waslarger than G0 and both moduli increased with increasing fre-quency. Similar liquid-like behavior, as shown in Fig. 2a, was ob-served for the lower phase of the phase-separated OGL110/Na-CASmixtures that corresponded to the bi-phasic zone of the diagram, atwhich both phases were in the liquid state (Fig. 1a). On the otherhand, for the lower phases of OGL110/Na-CAS and OGL35/Na-CASsystems, characterized as gels by macroscopic observation, the

mechanical spectra were typical of elastic gels; G0 was greater thanG00, and both moduli became almost independent of frequency(Fig. 2b and c).

The phase separation behavior of mixed oat b-glucan/pullulanaqueous dispersions also showed that above a certain b-glucan

0.01

0.1

1

10

100

1000

0.1 10Frequency (Hz)

a

0.01

0.1

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100

1000b

0.01

0.1

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G″ (upper)G″ (lower)

G″ (upper)G″ (lower)

G′ (initial)G′ (upper)G′ (lower)

G′,

G″

(P

a)

Fig. 2. Mechanical spectra (strain 0.1%, 20 �C) of the initial mixture and the upper andlower phases resulting after centrifugation (900g� 2 h) following 18 h aging at 20 �Cof oat b-glucans/sodium caseinate aqueous mixtures: OGL110 (3.1%)/Na-CAS (4.1%) (a),OGL110 (4.4%)/Na-CAS (3.8%) (b), and OGL35 (3.3%)/Na-CAS (2.4%) (c); compositions ofthe separated phases are given in Table 1. OGL110 and OGL35: oat b-glucans withapparent molecular weights of 110 and 35�103, respectively; Na-CAS: sodiumcaseinate; G0: elastic modulus; G00: loss modulus.

0.01

0.1

1

10

100

1000

0.1Frequency (Hz)

b

0

1

2

3

4

5

6

0 4 6 8

PULL (% w/w)

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L35 (%

w

/w

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Two phases (upper liquid; lower gel)One liquid phase

a

10

1 2 3 5 7

G′,

G″

(P

a)

1

G′ (initial)G′ (upper)G′ (lower)

G″ (initial)G″ (upper)G″ (lower)

Fig. 3. Bulk phase separation of oat b-glucans/pullulan aqueous dispersions aftercentrifugation (900g� 2 h) following 18 h aging at 20 �C; the points represent theinitial composition of the biopolymer mixtures (a). Mechanical spectra (strain 0.1%,20 �C) of the initial mixture OGL35 (3.2%)/PULL (2.6%) and of the upper and lowerphases; compositions of the separated phases are given in Table 1 (b). OGL35: oat b-glucans with apparent molecular weight of 35�103; PULL: pullulan; G0: elasticmodulus; G00: loss modulus.

A. Lazaridou, C.G. Biliaderis / Food Hydrocolloids 23 (2009) 886–895 889

concentration, there is incompatibility between the two poly-saccharides (Fig. 3a); following phase separation, the upper phaseremained liquid, whereas the lower phase turns into a gel (Fig. 3b).

The appearance of aqueous dispersions of oat b-glucans mixedwith sodium caseinate or pullulan after aging and centrifugation isdepicted in Fig. 4. For the zone of the diagrams where bulk phaseseparation was not observed, the mixtures preserved their trans-parent homogeneous liquid appearance, as shown in a representa-tive sample in Fig. 4a. In phase-separated systems, the two phaseswere visually distinguishable (Fig. 4b,c,e and f) with the upperphase being translucent and the lower phase opaque. As expected,in the cases that the lower phase was in a gel state (Fig. 4c,e and f)or when one gel phase appeared after aging (in OGL110/Na-CASmixtures, Fig. 4d) turbidity in these phases was evident, showingthe presence of aggregated structures. However, even for the bi-phasic OGL110/Na-CAS systems, at which the lower phase was

liquid-like (Fig. 4b), this phase also appeared turbid compared withits respective clear upper phase; in these cases, the lower phasewas a highly viscous fluid. This zone on OGL110/Na-CAS diagramsseemed to be an intermediate situation between mixtures with lowb-glucan concentration that appeared miscible and phase-sepa-rated systems containing high b-glucan levels, at which the lowphase exhibited a gel-like behavior (Fig. 1a). Cloudiness in the lowerliquid-like phase of this intermediate zone (Fig. 4b) suggests someintermolecular associations. This fact seems to be supported byrheological measurements, which showed an enhancement of G0

for this phase, compared to the values obtained for the respectiveinitial mixtures (Fig. 2a). Nevertheless, these molecular interactionsare not strong enough to form an extensive cross-linked gel net-work structure; definitely, a higher b-glucan concentration in themixture is required for gel formation in the lower phase, as clearlyindicated in Fig. 1a.

Fig. 4. Phase behaviour of oat b-glucans/sodium caseinate and oat b-glucans/pullulan aqueous dispersions: OGL110 (1.3%)/Na-CAS (2.6%) (a), OGL110 (2.8%)/Na-CAS (5.1%) (b),OGL110 (3.1%)/Na-CAS (5.7%) (c), OGL110 (3.5%)/Na-CAS (6.6%) (d), OGL35 (3.2%)/Na-CAS (1.6%) (e) and OGL35 (3.3%)/PULL (1.5%) (f); all photographs were taken after centrifugation(900g� 2 h) following 18 h aging at 20 �C of the mixtures, except photograph (d) which was taken after the aging process without centrifugation. The composition of the upper andlower phases for the phase-separated systems is given in Table 1. OGL110 and OGL35: oat b-glucans with apparent molecular weights of 110 and 35�103, respectively; Na-CAS:sodium caseinate; PULL: pullulan.

A. Lazaridou, C.G. Biliaderis / Food Hydrocolloids 23 (2009) 886–895890

For all aqueous mixtures of oat b-glucans with sodium ca-seinate or pullulan, even a very low concentration (w0.2%) of thesecond hydrocolloid was adequate for the system to exhibit im-miscibility (Figs. 1 and 3a). The concentration of b-glucans re-quired for inducing phase separation after aging andcentrifugation was strongly dependent on the molecular weight ofthe b-glucan preparation. For the high molecular weight b-glucan(OGL110) this concentration was also dependent on the sodiumcaseinate concentration in the initial mixtures (Fig. 1a). With in-creasing Na-CAS concentration the concentration of OGL110, atwhich incompatibility between the two hydrocolloids occurred,decreased in a sigmoidal manner. Thus, under the adopted ex-perimental conditions, at low (<2% w/w) protein levels, a rela-tively high b-glucan concentration, about 4% (w/w), is required toyield a phase-separated system. In the range of 2–5% (w/w)protein concentration, the b-glucan concentration required forimmiscibility dropped to 1% (w/w). On the other hand, with thetwo lower molecular weight b-glucans (OGL65 and OGL35) theboundary zone for a single liquid phase was relatively insensitiveto the concentration of the second polymeric constituent (Na-CAS,PULL); Figs. 1b, c and 3a versus Fig. 1a.

For the systems exhibiting two distinct layers, compositionalanalysis of the initial mixtures and the separated phases showeda slight increase in concentration of sodium caseinate or pullulan inthe upper phase (Table 1), less than 10%, and a larger increase inconcentration of b-glucans in the lower phase, in the range of w5–110% (Table 1, Fig. 5). The concentration increase of b-glucans in thelower phase was dependent on the initial hydrocolloid composition(Fig. 5); generally, a greater increase was found for mixtures withlow b-glucan and high sodium caseinate or pullulan concentration.The volume proportions of the two phases were also dependent onpolymeric composition (Fig. 6); a greater volume fraction for thelower phase was generally found for mixtures of high initial b-glucan concentration.

4. Discussion

The phase (state) diagrams as well as the rheological datashowed a concurrence of phase separation and gelation phenom-ena in mixed b-glucans/sodium caseinate and b-glucans/pullulanaqueous dispersions. Phase separation processes exhibit greatercomplexity when there is competition between polymer demixingand other events, such as gelation (Sciortino et al., 1993). Comparedto the well-documented gelling ability of b-glucans, pullulan isa non-gelling polysaccharide and sodium caseinate, at low con-centrations, neutral pH, and room temperature, as employed in thisstudy, is unlikely to form network structures. Manoj et al. (1996)using dynamic rheometry indicated that 17.5% sodium caseinatedispersions at neutral pH and at 5 �C exhibit a liquid-like behavior;only with more concentrated dispersions (e.g. 30%) the rheologicalresponses were typical of a gel material. Instead, oat b-glucanaqueous dispersions can gel at room temperature depending on theconcentration and the molecular weight of the polysaccharide(Lazaridou et al., 2003a). When dilute b-glucan aqueous dispersions(OGL110, 3.1% w/w and OGL35, 2.1% w/w) were aged (20 �C� 18 h)and centrifuged (900g� 2 h) no gelation was observed (data noshowed). However, in the presence of sodium caseinate and pul-lulan the mixed b-glucan/hydrocolloid dispersions resulted ina phase-separated system with the lower phase being in a gel state(Figs. 1 and 3a); the latter phase was enriched in b-glucan (Table 1and Fig. 5). It seems that phase separation phenomena induceintermolecular associations of b-glucan chains and thereby gelnetwork formation due to increasing polysaccharide concentrationin the lower phase. It is well known that the addition of anotherhydrocolloid (gel-forming or not) to a solution of a gel-formingpolymer decreases the critical concentration and increases the rateof gelation due to polymer immiscibility (Sciortino et al., 1993;Tolstoguzov, 1995, 2003). Manoj et al. (1996) have reported similarbehavior in sodium caseinate/maltodextrin mixed systems. They

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A. Lazaridou, C.G. Biliaderis / Food Hydrocolloids 23 (2009) 886–895 891

have suggested a preferential hydration of sodium caseinate thatraises the effective concentration of maltodextrin in its respectivemicrodomains above the minimum critical gelling concentration ofthe starch hydrolyzate. Similarly, our data showed a decrease in therequired b-glucan concentration at which this polysaccharide gel-led in the mixtures with increasing concentration of the secondhydrocolloid (Figs. 1 and 3a); this behavior was more pronouncedfor the OGL110/Na-CAS system (Fig. 1a). In agreement with thesefindings, Kontogiorgos et al. (2006), studying the network proper-ties of the phase-separated sodium caseinate/barley b-glucansmixed gels, found that the sodium caseinate phase exhibits anabout 50% higher water holding capacity than its polysaccharidecounterpart.

Immiscible biopolymer mixtures showing similar behavior tothe mixed systems of the present study, i.e. aggregation and gela-tion processes caused by phase separation, have been previouslyreported (Garnier et al., 1995). Mixed dextran/locust bean gumaqueous dispersions exhibited bulk phase separation with a liquidlower phase and an upper gel phase. The gel structure formationwas ascribed to self-association of the galactomannan chains due tothe significant concentration increase of the locust bean gum. Forgelatin/maltodextrin systems at 45 �C, at which the individualpolymers exist as stable disordered coils, it was demonstrated thatthe presence of gelatin drives the self-association and ordering ofthe maltodextrin chains (Kasapis et al., 1993). For amylose/xanthansystems, the thermodynamic incompatibility between the twopolysaccharides was also used to explain the increase of rate ofamylose aggregation, as measured by turbidity tests, and themacroscopic precipitation of the amylose aggregates as well(Mandala, Michon, & Launay, 2004). Despite that sodium caseinate/xanthan mixtures at neutral pH and room temperature did notexhibit bulk phase separation, confocal laser scanning microscopy(CLSM) revealed thermodynamic incompatibility for these mix-tures under certain conditions; the presence of sodium caseinateresulted in ‘thread-like’ xanthan-rich regions probably due to self-association of the xanthan chains.

For mixtures containing sodium caseinate up to approximately4% (w/w), much higher levels of OGL110 were required to inducephase separation than those of OGL65 and OGL35 (Fig. 1). Previousfindings for pure b-glucan dispersions showed that the gelationrate decreased with increasing molecular weights of the poly-saccharide (Lazaridou et al., 2003a). It would appear that theincreased tendency for gelation of the low molecular weight b-glucans accelerate demixing of the two hydrocolloids. In otherstudies, it has been demonstrated that the gelation process caninduce the phase separation phenomena (Alves et al., 1999, 2000;Kasapis et al., 1993; Loren et al., 2001; Tromp et al., 1995). Molecularordering and chain aggregation events can drive phase separation,probably due to the increase of the effective molecular weight(Norton & Frith, 2001). Similarly, a displacement of the binodaltowards lower concentrations of biopolymers has been shown forgelatin/locust bean gum and for gelatin/dextran systems, concur-rent with a transition of gelatin from a molecularly dispersed stateto a molecular aggregated system, e.g. with decreasing temperaturebelow the critical gelation temperature of gelatin (Alves et al., 1999,2000; Tromp et al., 1995). Furthermore, for gelatin/maltodextrinsystems, Loren and Hermansson (2000) showed that conforma-tional ordering of gelatin is possible to initiate demixing both belowand above the gelation temperature of gelatin by a small change incomposition of the initial mixture. In a later study, Loren et al.(2001) using optical rotation and turbidity measurements in gela-tin/maltodextrin systems showed that the degree of gelatin or-dering appeared to determine the initiation of phase separation;a higher degree of gelatin ordering shifted the binodal towardslower gelatin and maltodextrin concentrations. All these findingsare in accord with the observation that b-glucans with a higher

01 2

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Na-CAS (%

w/w

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11.522.533.54

OGL110 (% w/w)

1.522.533.54

OGL35 (% w/w)

00

2020

4040

6060

8080

100100

120 120

Co

ncen

tratio

n in

crease o

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110 (%

)

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ncen

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35 (%

)

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ncen

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crease o

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)

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crease o

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b

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Na-CAS (%

w/w

)

1

1.522.533.54

OGL35 (% w/w)1 0

1 23 4 5 6 7

PULL (% w

/w)

Fig. 5. Three-dimensional plots of percentage increase of b-glucan concentration in the lower phase of phase-separated oat b-glucans/sodium caseinate and oat b-glucans/pullulanaqueous dispersions as a function of concentration of the individual hydrocolloids in the initial mixtures; open symbols represent phase-separated systems, where both phaseswere liquid, whereas filled symbols represent the phase-separated systems, in which the upper phase was liquid and the lower phase was gel. Phase separation was accomplishedby centrifugation (900g� 2 h) following 18 h aging at 20 �C of the dispersions. OGL110 and OGL35: oat b-glucans with apparent molecular weights of 110 and 35�103, respectively;Na-CAS: sodium caseinate; PULL: pullulan.

A. Lazaridou, C.G. Biliaderis / Food Hydrocolloids 23 (2009) 886–895892

01 2 3 4 5 6 7

Na-CAS (%

w/w

)

01 2 3 4 5 6 7

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w/w

)

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4 5 6 7

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/w)

11.522.533.54

OGL110 (% w/w)

1.522.533.54

OGL35 (% w/w)1

1.5 122.533.54

OGL35 (% w/w)

0

0

2020

4040

6060

80 80

100 100

Lo

wer p

hase (%

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hase (%

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hase (%

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wer p

hase (%

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Lo

wer p

hase (%

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a

b

c

Fig. 6. Three-dimensional plots of volume percentage of the lower phase in phase-separated oat b-glucans/sodium caseinate and oat b-glucans/pullulan aqueous dispersions asa function of concentration of the individual hydrocolloids in the initial mixtures; open symbols represent phase-separated systems, where both phases were liquid, whereas filledsymbols represent the phase-separated systems, in which the upper phase was liquid and the lower phase was a gel. Phase separation was accomplished by centrifugation(900g� 2 h) following 18 h aging at 20 �C of the dispersions. OGL110 and OGL35: oat b-glucans with apparent molecular weights of 110 and 35�103, respectively; Na-CAS: sodiumcaseinate; PULL: pullulan.

A. Lazaridou, C.G. Biliaderis / Food Hydrocolloids 23 (2009) 886–895 893

A. Lazaridou, C.G. Biliaderis / Food Hydrocolloids 23 (2009) 886–895894

tendency for ordering (i.e. low molecular weight preparations)reduced the polysaccharide concentration required for inducingphase separation (Figs. 1 and 3a).

The experimental data of this study showed relatively low in-creases of b-glucan concentration in the lower phase, up to onefold(Fig. 5), and minor enrichment of the sodium caseinate or pullulanin the upper phase (Table 1). Most likely, aggregation of b-glucanchains dramatically increases the viscosity of the system and hin-ders the mobility of components. On the other hand, the enrich-ment of b-glucans in the lower phase was augmented in systemshaving low initial concentrations of b-glucans in combination withhigh levels of sodium caseinate or pullulan (Fig. 5). For dextran/locust bean gum immiscible systems, showing similar behavior tothe mixtures described in the present study, an extremely highconcentration increase of galactomannans in the upper gel phase,reaching 200 and up to 500%, and the absence of this poly-saccharide from the lower phase was found; nevertheless, the totaldiffusion of the dextran towards the lower liquid phase wasimpeded by gelation of locust bean gum (Garnier et al., 1995).

As a result of limited mobility, there was also formation ofa homogeneous gel-like phase in OGL110/Na-CAS for mixtures withhigh b-glucan initial concentrations and aged at 20 �C for 18 h (Figs1a and 4d). In this case, phase separation was ‘arrested’ by gelation.For gelatin/dextran (Owen & Jones, 1998; Tromp et al., 1995; Tromp& Jones, 1996) and gelatin/maltodextrin (Alevisopoulos et al., 1996;Butler, 2002; Loren et al., 1999, 2001; Loren & Hermansson, 2000)systems at which gelation can take place simultaneously withphase separation, the immobility of the gelling component inhibitsthe separation process, depending on the gelation rate relatively tothe separation rate; the latter relationship, in turn, depends onpolymer concentration, temperature and quenching cooling rates,and further determines the system microstructure. Other similaraqueous biopolymer mixtures were found to exhibit only a micro-scopic and not a bulk phase separation (Bourriot et al., 1999; Trompet al., 1995).

A comparison between OGL35/Na-CAS and OGL35/PULL sys-tems with a similar initial concentration of b-glucan and at equiv-alent initial concentrations of sodium caseinate and pullulan, theOGL35/Na-CAS mixtures showed smaller enrichment of b-glucansin the lower phase than that observed for the respective OGL35/PULL mixed dispersions (Fig. 5b and c). This observation reflectsslower phase separation kinetics of the former mixtures and couldbe attributed to the higher apparent viscosity values that weredisplayed by sodium caseinate than the pullulan dispersions at anequivalent polymer concentration (data not shown). The lowermobility of the OGL35/Na-CAS systems, compared to those withpullulan, was also evident from the higher volume fraction values(%) of the lower phase in OGL35/Na-CAS mixtures (80–90%) versus40–60% of the OGL35/PULL at b-glucan concentrations of w3.0–3.5% (Figs 6b,c and 4e,f). At high b-glucan levels, a more viscousmedium, caused by chain aggregation, seems to delay the phaseseparation events and results in a higher volume fraction of thelower phase (Fig. 6). Similarly, in casein/amylopectin systems ata fixed initial casein concentration, an increase of the height of thesediment formed by the protein-rich phase was observed withincreasing amylopectin concentrations (de Bont et al., 2004).

5. Conclusions

Mixed oat b-glucans/sodium caseinate and oat b-glucans/pul-lulan aqueous dispersions showed thermodynamic in-compatibility, as indicated by the macroscopic phase separation ofthese systems. The molecular weight of oat b-glucans and initialcomposition of the mixtures were found to influence their im-miscibility. In these systems, phase separation seems to coexist andcompete with chain aggregation–gelation phenomena. Phase

separation can drive and accelerate formation of gel b-glucanenriched network structures; in many cases of the phase-separatedsystems, the lower phase was found in a gel state. Conversely,gelation appeared to induce and promote phase separation.Therefore, the immiscibility between oat b-glucans and otherhydrocolloids was found to be affected by the gelling tendency ofb-glucans, which generally increases with decreasing molecularsizes of the polysaccharide chains. Thus, the concentration ofb-glucans required to induce bulk phase separation was lower forb-glucans of lower molecular weight than with high molecularweight samples. Bulk phase separation also seemed to be ‘arrested’by gelation. Moreover, the kinetic entrapment of the componentsinto a highly viscous medium, appeared to influence the compo-sition and volume fraction of the individual phases, resulting in anincomplete phase separation. Overall, phase diagrams of suchsystems are diagrams of state rather than typical phase diagramsestablished under equilibrium conditions.

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